Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells

Netelenbos, Tanja; Dräger, A.M.; Hof, Bert van het; Kessler, Floortje L.; Delouis, Claude; Huijgens, Peter C.; Born, Jacob van den; Dijk, W. van

doi:10.1016/s0301-472x(01)00653-1

Cited by 40 publications

(37 citation statements)

References 50 publications

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“…Human BMEC cell lines 4LHBMEC 28 (used in most experiments), TrHBMEC 10 and HBMEC60 30 (kindly provided by Dr CE van der Schoot, CLB, Amsterdam, The Netherlands) were cultured in endothelial cell culture medium consisting of medium 199, 10% new born calf serum, 1% glutamine (Gibco, Grand Island, NY, USA), 10% human pooled serum (CLB, or BioWhittaker, Walkersville, MD, USA), 0.15 mg/ml crude endothelial cell growth factor and 5 IU/ml heparin at 37°C and 5% CO 2 in six-well plates coated with fibronectin (kindly provided by Dr JA van Mourik (CLB)) and passaged twice a week by detachment by trypsin-EDTA (BioWhittaker). HUVEC were isolated as described by Jaffe et al 31 from umbilical cords obtained from the department of Gynecology and Obstetrics (Vrije Universiteit Medical Center, Amsterdam, The Netherlands) after informed consent.…”

Section: Cell Culturesmentioning

confidence: 99%

“…28 After detachment from culture plates with EDTA, 200 000 endothelial cells were incubated in 50 l RPMI/0.05% BSA with 0.5 ng 125 I-SDF-1␣ for 2 h on ice in the presence of various GAGs at a concentration of 200 g/ml or with medium alone. Cells were centrifuged twice at 700 g at 4°C for 5 min and cell pellets were resuspended in 500 l RPMI/0.05% BSA.…”

Section: Competition Of 125 I-sdf-1 Binding To 4lhbmec With Gagsmentioning

confidence: 99%

“…PGs were isolated from 4LHBMEC by a method previously described, 28 with some modifications. In brief, confluent cultures of 4LHBMEC and HUVEC in T175 culture flasks were metabolically labeled with 22 uCi/ml 3 H glucosamine (NEN, 48.3 Ci/mmol) for 16 h at 37°C in MEM (Gibco) with 2% human serum.…”

Section: Isolation Of Pgs and Hs-gags From 4lhbmec And Huvecmentioning

confidence: 99%

“…In contrast, and in accordance with literature, 40,41 the binding of the protein bFGF to heparin was more pronounced. To study whether intact PGs, ie GAGs attached to their core proteins, would have a better binding capacity towards SDF-1, SDF-1 was immobilized in a microplate and the total fraction of PGs (HSPGs and CS/DSPGs together (ratio approximately 1:1) 28 was allowed to bind. Similar results were obtained as in the filter binding assay, ie weak binding of heparin and no binding with isolated PGs, whereas in coated bFGF-wells both heparin and PGs were able to bind (not shown).…”

Section: No Binding Of Sdf-1 To Isolated Endothelial Hs-gags and Pgsmentioning

confidence: 99%

“…We reported recently that cell-associated HS-GAGs from the BMEC cell line 4LHBMEC contained higher sulfated domains compared to similar GAGs from human umbilical vein endothelial cells (HUVEC). 28 Furthermore, it was found that 4LHBMEC bound more 125 I-SDF-1␣ compared to HUVEC per cell. This binding could be inhibited by competition with heparin, O-sulfated heparin (N-desulfated, N-acetylated heparin) and to a lesser extent by N-sulfated heparin (completely desulfated, N-sulfated heparin), suggesting a role for an O-sulfated motif in endothelial HS-GAGs in the binding of SDF-1.…”

Section: Introductionmentioning

confidence: 98%

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Section: Cell Culturesmentioning

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Section: Competition Of 125 I-sdf-1 Binding To 4lhbmec With Gagsmentioning

confidence: 99%

Section: Isolation Of Pgs and Hs-gags From 4lhbmec And Huvecmentioning

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Section: No Binding Of Sdf-1 To Isolated Endothelial Hs-gags and Pgsmentioning

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confidence: 98%

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Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells

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Laser‐induced fluorescence as a powerful detection tool for capillary electrophoretic analysis of heparin/heparan sulfate disaccharides

Militsopoulou

Lecomte

Bayle

et al. 2003

Biomedical Chromatography

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In quest for high sensitivities necessary for determining the disaccharide composition of heparin/heparan sulfate present in trace amounts in biologic samples, an ultrahighly sensitive capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection was developed. Heparin/heparan sulfate-derived Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone and resolved by a reversed-polarity CE method. Estimation of the limit of detection in concentration term and limit of quantitation showed that LIF detection of AMAC-derivatives of Delta-disaccharides resulted in 27-744 times higher sensitivity as compared to those detected by UV at 255 nm. These data suggest that CE-LIF is a powerful tool to quantify minute amounts of heparin/heparan sulfate disaccharides.

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Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection

et al. 2008

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This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan disaccharide analysis from complex biological samples. In the process of applying capillary electrophoresis with laser-induced fluorescence to glycosaminoglycan (GAG) disaccharide analysis in biological samples, we have made improvements to existing methods. These include (1) optimization of reductive amination conditions, (2) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization and, (3) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate disaccharides or eleven of twelve standard heparin/HS disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1% to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem mass spectrometry. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/HS and CS/DS disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.

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Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells

Cited by 40 publications

References 50 publications

Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells

Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells

Laser‐induced fluorescence as a powerful detection tool for capillary electrophoretic analysis of heparin/heparan sulfate disaccharides

Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection

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